Mutant mono-oxygenase cytochrome P-450 cam

ABSTRACT

A mutant of the mono-oxygenase cytochrome P-450 cam  in which the tyrosine residue at position 96 and/or the cysteine residue at positon 334 is replaced by the residue of any amino acid except phenylalanine.

This application is the National Stage of International Application No.PCT/GB95/02588, filed Nov. 2, 1995.

The present invention relates to a mutant of the mono-oxygenasecytochrome P-450_(cam) and method of oxidising certain organic compoundswith the mutant.

BACKGROUND OF THE INVENTION

Mono-oxygenases catalyse the selective oxidation of non-functionalisedhydrocarbons using oxygen¹, and are therefore of great interest forpotential use in organic synthesis. However, progress in this area hasbeen hampered by the difficulty in isolating sufficient quantities ofenzyme and the associated electron-transfer proteins. Despite theavailability of amino acid sequences of more than 150 differentcytochrome P-450 mono-oxygenases, to date structural data of only threeare available²,3,4, and few have been successfully over-expressed inbacterial systems⁵.

One cytochrome P-450 mono-oxygenase, which is soluble and can beexpressed in sufficient quantities, is the highly specific P-450_(cam)from P.putida which catalyses the regio- and stereo-selectivehydroxylation of camphor (1) to 5-exo-hydroxycamphor⁶. The highresolution crystal structure of P-450_(cam) has been determined², andsince the mechanism of action of this bacterial enzyme is believed to bevery similar to that of its mammalian counterparts, it has been used asa framework on which models of mammalian enzymes are based.

The nucleotide sequence and corresponding amino acid sequence ofP-450_(cam) have been described⁵. The location of an active site of theenzyme is known and structure-function relationships have beeninvestigated¹³, 14. Mutants of P-450_(cam) have been described, at the101 and 185 and 247 positions¹⁵, and at the 87 position¹⁶. A mutant inwhich tyrosine 96 has been changed to phenyl alanine-96 has beendescribed¹²,17,18. But in all these cases the papers report effects ofthe mutations on the mechanisms of known oxidation reactions. There isno teaching or suggestion that mutation might be used to providebiocatalysts for oxidation of different substrates.

SUMMARY OF THE INVENTION

In an attempt to find new biocatalysts, we have initiated a projectwhich aims to redesign the active site of P-450_(cam), such that it isable to carry out specific oxidations of organic molecules which are notsubstrates for the wild-type protein. Our initial aim was to incorporatean "aromatic pocket" into the P-450_(cam) active site, which wouldencourage the binding of substrates containing aromatic side-chains.

In addition, a surface residue remote from the active site wasidentified (cysteine-334) with effects on protein handling andstability. The cysteine is responsible for unwanted dimerisation of theprotein during purification and an alanine residue was thereforesubstituted for the cysteine in order to improve both of theseproperties.

The three dimensional structure of P-450_(cam) shows the active site toprovide close van der Waals contact with the hydrophobic groups ofcamphor as shown in FIG. 1. Three aromatic residues (Y96, F87 and F98)are grouped together and line the substrate binding pocket, with ahydrogen bond between tyrosine 96 and the camphor carbonyl oxygenmaintaining the substrate in the correct orientation to ensure theregio-and stereo-specificity of the reaction. Replacement of any ofthese aromatic residues with a smaller, hydrophobic non-aromaticside-chain could provide the desired "aromatic pocket".

Molecular modelling was used to investigate the likely effects of pointmutations to the three aromatic residues. The program GRID⁷ was used tocalculate an energy of interaction between an aromatic probe andpossible mutants of cytochrome P-450_(cam) where these residues werechanged to alanine (F87A, Y96A and F98A). The results were then examinedgraphically using the molecular modelling package Quanta⁸.

The mutant F98A appeared to have the strongest binding interactionwithin the active site cavity accessible to the aromatic probe, withthat of Y96A being slightly smaller, and that of F87A beingsubstantially less. It was decided in the first instance to mutatetyrosine 96 to alanine as it is more central to the binding pocket,whereas phenylalanine 98 is in a groove to one side. Also, removal oftyrosine 96 should decrease the specificity of the enzyme towardscamphor due to the loss of hydrogen bonding to the substrate.

According to one aspect of the present invention a mutant of themono-oxygenase cytochrome P-450_(cam) is provided in which the tyrosineresidue at position 96 and/or the cysteine residue at position 334 isreplaced by the residue of any amino acid except phenylalanine.

According to another aspect of the present invention a mutant of themono-oxygenase cytochrome P-450_(cam) is provided in which the tyrosineresidue at position 96 and/or the cysteine residue at position 334 isreplaced by another amino acid residue, which mutant has the property ofcatalysing the oxidation of any one of the following:- polycyclicaromatic hydrocarbons, linear or branched alkanes, diphenyl and biphenylcompounds including halogenated variants of such compounds andhalogenated hydrocarbons.

According to yet another aspect of the present invention a method isprovided of oxidising a compound selected from a polycyclic aromatichydrocarbon, a linear or branched alkane, a diphenyl or biphenylcompound including a halogenated variant of such a compound or ahalogenated hydrocarbon, the method comprising contacting the selectedone of the compounds under oxidising conditions with mono-oxygenasecytochrome P-450_(cam) in which the tyrosine residue at position 96and/or the cysteine residue at position 334 is replaced by another aminoacid residue.

Preferably the amino acid is selected from any one of the following:alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,proline, serine, threonine, tryptophan, tyrosine and valine except thatin the case of the tyrosine residue at position 96, the amino acid isnot tyrosine and in the case of the cysteine residue at position 334,the amino acid is not cysteine.

The amino acid which replaces tyrosine at position 96 is convenientlyone of the small hydrophobic amino acids, e.g. alanine, glycine, valine,leucine or isoleucine, with alanine being preferred as exemplifiedbelow.

Alternatively the amino acid replacing tyrosine at position 96 may beone of the charged amino acids, e.g. a negatively charged acid such asaspartic acid or glutamic acid for hydrogen bonding to a positivelycharged substrate; or a positively charged compound such a lysine,arginine or histidine for hydrogen bonding to a negatively chargedsubstrate which are not members of the camphor family.

The mutation at position 96 is believed to be the key which enables themutant enzymes to catalyse the oxidation of a relatively wide range oforganic substrates. Other amino acids adjacent to the active site of theenzyme may also be mutated in order to change the shape and specificityof the active site. These other amino acids include those at positions87, 98, 185, 244, 247, 295 and 297. It is envisaged that the amino acidat one or more of these positions may be replaced by: a smallhydrophobic amino acid so as to enlarge the active site; or a largehydrophobic amino acid so as to reduce the size of the active site; orby an amino acid having an aromatic ring to bond to a correspondingaromatic ring of a substrate.

Regarding the oxidising reactions, the conditions are described in theliterature references attached. The enzyme system typically includesputidaredoxin and putidaredoxin reductase together with NADH asco-factors in addition to the mutant enzyme. Various classes of organiccompounds are envisaged:

i) The organic compound is an aromatic compound, either a hydrocarbon ora compound used under conditions in which it does not inactivate ordenature the enzyme. Since the mutation has been effected with a view tocreating an aromatic-binding pocket in the surface of the enzyme, themutant enzyme is capable of catalysing oxidation of a wide variety ofaromatic compounds. Oxidation of example aromatic and polyaromaticcompounds is demonstrated in the experimental section below and isbelieved very surprising given that the wild-type enzyme catalyses theoxidisation of only members of the camphor family.

ii) The organic compound may be a hydrocarbon, e.g. aliphatic oralicyclic, carrying a functional group. An aromatic protective group isadded to the functional group prior to the oxidation reaction andremoved from the functional group after the oxidation reaction. Asuitable aromatic group is a phenyl group. The aromatic protection groupis expected to hold the substrate in place. Thus the protecting groupserves two purposes: firstly it makes the substrate more hydrophobic andhence increases binding to the hydrophobic enzyme pocket; secondly itholds the substrate in place at the active site. Thus, with the correctaromatic protection group, both regio-and stereo-selective hydroxylationof the substrate may be achieved. The example of cyclohexylbenzene isdescribed in the experimental section below.

Examples of monofunctionalised hydrocarbons are cyclohexyl, cyclopentyland alkyl derivatives (Scheme 1). The oxidation products of thesecompounds are valuable starting materials for organic synthesis,particularly when produced in a homochiral form. A range of aromaticprotecting groups are envisaged, e.g. benzyl or naphtyl ethers andbenzoyl or naphthoyl esters and amides (Scheme 1). Of interest are alsobenzoxazole groups as carboxyl protecting groups and N-benyl oxazolidinegroups as aldehyde protecting groups. Both can be easily cleaved afterthe enzymatic oxidation and have previously been described in theliterature for the microbial oxidations of aldehydes and acids.

iii) The organic compound is a C5 to C12 aliphatic or alicyclichydrocarbon. Oxidation of cyclohexane and linear hydrocarbons isdemonstrated in the experimental section below and once again it isbelieved quite surprising given that the wild-type enzyme catalyses theoxidation of only members of the camphor family.

iv) The organic compound is a halogenated aliphatic or alicyclichydrocarbon. Oxidation of lindane (hexachlorocyclohexane) is alsodescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a showing that the three dimensional structure of P-450_(cam)shows the active site to provide close van der Waals contact with thehydrophobic groups of camphor. FIG. 2 is a gas chromatograph ofdiphenylmethane (A) and hydroxylated product formed following incubationwith P-450_(cam) Y96A mutant.

Based on the above considerations, mutant proteins were constructedwhich contained alanine, lysine, valine, or phenylalanine instead oftyrosine at position 96 (Y96). Additional mutants were constructed inwhich these active site replacements were combined with the surfacemutation of cysteine at position 334 to alanine. Lastly several activesite mutations and the surface mutation were combined in one protein toconstitute a multiple mutant enzyme. The genes encoding cytochromeP-450_(cam), and its natural electron-transfer partners puridaredoxinand putidaredoxin reductase, were amplified from the total cellular DNAof P. Putida using the polymerise chain reaction (PCR). The expressionvector/E. coli host combinations employed were pRH1091⁹ in strain JM109for P-450_(cam), pUC 118 in strain JM109 for putidaredoxin, and pGLW11in strain DH5^(oc) for putidaredoxin reductase. Oligonucleotide-directedsite-specific mutagenesis was carried out using an M13mp19 subclone bythe method of Zoller and Smith¹⁰, and mutant selection was by the methodof Kunkel¹¹.

The mutant Y96A was shown to catalyse the hydroxylation of camphor (1),although compared to the wild-type enzyme the reaction was lessselective, similar to that reported for the mutant Y96F¹². This decreasein selectivity can be attributed to the loss of the hydrogen bondbetween Y96 and camphor. The properties of wild-type and Y96A proteinswere further investigated with a variety of binding and activity assays.

Binding of potential substrates was investigated by spectroscopicmethods. The wild-types enzyme in the absence of substrate is in the6-co-ordinated, low-spin form with a weakly bound water occupying thesixth co-ordination site, and shows a characteristic Soret maximum at391 nm. Binding of the substrate analogues adamantanone (2), adamantane(3) and norbornane (4) also fully converted the haem to the high-spinform. However, diphenylmethane (5) did not give a shift in theabsorption spectrum.

The Y96A mutant, while giving the same results for compounds (3) and(4), was not fully converted to the high-spin form even when (1) and (2)were added in excess. Most interestingly however, and in contrast to thewild-type, Y96A showed partial conversion to the haem to the high-speedform with diphenylmethane, indicating binding of this compound to themutant protein.

As expected, the dissociation constants (K_(app)) for camphor andadamantanone are increased in Y96A. On the other hand, the K_(app)values for the hydrophobic substrates adamantane and norbornane arereduced, indicating that the enzyme pocket has become more selective forhydrophobic substrates. The greatest change in binding was obtained withdiphenylmethane, which bound poorly to wild-type protein, but showedgreatly enhanced affinity for the Y96A mutant (Table 1).

Once binding of diphenylmethane by the Y96A protein had beenestablished, catalytic substrate turnover was investigated. The mutantprotein was reconstituted with putidaredoxin and putidaredoxinreductase. Diphenylmethane (5) was added and the mixture was incubatedwith NADH and oxygen.

A solution containing 10 μM putidaredoxin, 2 μM putidaredoxin reductase,1 μM cytochrome P-450_(cam) mono-oyxgenase (wild-type or mutant) and 1mM diphenylmethane in 100 mM KC1, 20 mM KH₂ PO₄ pH7.4 was preincubatedat 25° C. in a shaker for 5 min. The enzymatic reaction was initiated byfirstly adding NADH to a total concentration of 2 mM. Further fouraliquots of NADH (to increase the NADH concentration by 1 mM each time)were added in intervals of 5 min and the reaction quenched after 30 minby adding 0.5 ml chloroform. The chloroform layer was analysed by gaschromatography.

Organic extracts of the crude incubation mixture were analysed by gaschromatography. Only one major new peak was detected by GC (see FIG. 2),which had the same retention time as an authentic sample ofpara-hydroxydiphenylmethane (6). The other aromatic hydroxylationproducts, the ortho and meta isomers, had different retention times.Further confirmation of the identity of the product as structure (6) wasprovided by mass spectrometry, which gave the correct mass peak at 184.

Using the above experimental techniques, the inventors have investigateda considerable number of organic compounds as substrates for both thewild-type P-450_(cam) enzyme and also the mutant version Y96A. Furtherwork has included mutants designated Y96V; Y96L; Y96F; C334A; thecombined mutant F87A, Y96G, F193A and the combined active site andsurface mutants of Y96A, C334A; Y96V, C334A; Y96L, C334A; Y96F, C334A;F87A, Y96G, F193A, C334A.

The results for Y96A are set out in Table 2, in which structurallyrelated molecules are grouped together. Those substrates where oxidationhas been demonstrated by means of NADH turnover are marked with a +sign.

Spin high/low: numbers shows the percentage of P-450 (OD₄₁₇ 0.2-0.4)converted from the low- to high-spin equilibrium state in the presenceof 200 μM test compound, in phosphate buffer (40 mM phosphate, 68 mMpotassium, pH 7.4). Spin state equilibrium is assessed with a UV/visspectrophotometer: low spin at OD₄₁₇ and high spin at OD392 nd; notdone.

Vs DTT: numbers show the percentage displacement of DTT (200 μM) boundto P-450 by competition with test compounds (200 μM) in phosphatebuffer. DTT binding to P-450 results in absorbance peaks at OD374 andOD₄₆₁, so displacement is measured with a UV/vis spectrophotometer.

Examples are included in Table 2(a) to 2(h) for each class of compoundsidentified in points (i) to (iv) above.

Reaction products for some substrate compounds have been purified byhigh performance liquid chromatography and identified by massspectroscopy, nuclear magnetic resonance, and/or co-elution. Table 3details the NADH consumption for oxidation of small linear, branched andcyclic hydrocarbons by the mutant Y96A, C334A. Table 4(a) to 4(h)details the product distributions for mutant and substrate combinationswhere this has been elucidated to date.

    __________________________________________________________________________    Scheme 1                                                                      Hydrocarbons                                                                           ##STR1##                                                                              ##STR2##                                                                              ##STR3##                                                                              ##STR4##                                                                              ##STR5##                             --Z     Protecting Group                                                      __________________________________________________________________________    --OH                                                                                   ##STR6##                                                                                 ##STR7##                                                  --NH.sub.2                                                                             ##STR8##                                                             --COOH                                                                                 ##STR9##                                                                                 ##STR10##                                                 --CHO                                                                                  ##STR11##                                                            __________________________________________________________________________

                  TABLE 1                                                         ______________________________________                                                            K.sub.app (μM).sup.a                                                       WT    Y96A                                                ______________________________________                                         ##STR12##        1       6.3     12                                           ##STR13##        2       12      28                                           ##STR14##        3       8.4     1.4                                          ##STR15##        4       330     92                                           ##STR16##        5       >1500.sup.b                                                                           73                                          ______________________________________                                         .sup.a Values are the average of two independent measurements using the       method of Sligar (S. G. Sligar, Biochemistry, 1976, 15, 5399-5406). The       value of K.sub.app is strongly dependent on the concentration of K.sup.+      in the buffer. At [K.sup.+ ] > 150 mM. K.sub.app for camphor is 0.6 μM     for both wildtype and Y96A. Data in this table were determined at [K.sup.     ] = 70 mM in phosphate buffer, pH 7.4, in order to avoid salting out of       substrates at higher ion concentrations.                                      .sup.b Saturation not reached.                                           

                                      TABLE 2(a)                                  __________________________________________________________________________                           Wild type Mutant Y96A                                                                             Wild type Mutant Y96A              P450cam-substrate Interactions                                                                       ΔSpin                                                                             ΔSpin                                                                             NADH      NADH                     Subgroup: 1-ring       high/low                                                                           Vs DTT                                                                             high/low                                                                           Vs DTT                                                                             turnover?                                                                           GC? turnover?                                                                           GC?                __________________________________________________________________________     ##STR17##  Benzene    --   --   --   --                                       ##STR18##  Toluene    --   --   30   30                                       ##STR19##  Ethylbenzene                                                                             --   --   40   40                                       ##STR20##  Styrene    --   --   30   30                                       ##STR21##  Cyclohexene                                                                              --    5   40   40                                       ##STR22##  1,3-Cyclohexadiene                                                                       nd   nd   nd   nd                                       ##STR23##  1,4-Cyclohexadiene                                                                       --    5   15   20                                       ##STR24##  Cyclohexane                                                                              --   --   60   60             +                         ##STR25##  Hexane     --   --   70   60             +                         ##STR26##  Methylcyclohexane                                                                        --   --   70   60                                       ##STR27##  (S)-(+)-Carvone                                                                          10   60   10   80                                      __________________________________________________________________________

                                      TABLE 2(b)                                  __________________________________________________________________________                                   Wild type                                                                              Mutant Y96A                                                                            Wild type                                                                            Mutant Y96A                                          ΔSpin                                                                            ΔSpin                                                                            NADH   NADH                  P450cam-substrate interactions high/    high/    turn-  turn-                 Subgroup: 2-ring, Naphthalene  low Vs DTT                                                                             low Vs DTT                                                                             over?                                                                             GC?                                                                              over?                                                                             GC?               __________________________________________________________________________     ##STR28##        Naphthalene  --  --   15  20                                 ##STR29##        1-Ethylnaphthalene                                                                         --  --    5  20                                 ##STR30##        2-Ethylnaphthalene                                                                         --  --   10  20                                 ##STR31##        2-Naphthylacetate                                                                          --   5   --   5                                 ##STR32##        1-Naphthylacetate                                                                          --   5   --   5                                 ##STR33##        1-Naphthylpropionate                                                                       --  20    0  20                                 ##STR34##        1-Naphthylbutyrate                                                                         --   5   --   5                                 ##STR35##        Naphthylphenylketone                                                                       --   5   --   5                                 ##STR36##        1,2-Dihydronaphthalene                                                                     5   20   30  90                                 ##STR37##        1,2,3,4-Tetrahydro  naphthalene                                                            5   10   40  40                                __________________________________________________________________________

                                      TABLE 2(c)                                  __________________________________________________________________________                                   Wild type                                                                              Mutant Y96A                                                                            Wild type                                                                            Mutant Y96A                                          ΔSpin                                                                            ΔSpin                                                                            NADH   NADH                  P450cam-substrate interactions high/    high/    turn-  turn-                 Subgroup: 2-ring, DPM          low Vs DTT                                                                             low Vs DTT                                                                             over?                                                                             GC?                                                                              over?                                                                             GC?               __________________________________________________________________________     ##STR38##        Diphenylmethane                                                                            --   5   45  nd          +  +                   ##STR39##        Diphenylether                                                                              10   5   20  50                                 ##STR40##        Benzophenone --  20   --  20                                 ##STR41##        Cyclohexylphenylketone                                                                     --  30   60  nd                                 ##STR42##        Phenylbenzoate                                                                             --   5   --  --                                 ##STR43##        N-Phenylbenzylamine                                                                             5   45  nd                                 ##STR44##        Bibenzyl     --  --   55  55                                 ##STR45##        cis-Stilbene --  20   40  50                                 ##STR46##        Biphenyl     --  20   --  90                                 ##STR47##        Cyclohexylbenzene                                                                          20  20   80  nd                                 ##STR48##        trans-Stilbene                                                                             --  --   --  --                                 ##STR49##        Benzylether  --   5   55  nd                                __________________________________________________________________________

                                      TABLE 2(d)                                  __________________________________________________________________________                                   Wild type                                                                              Mutant Y96A                                                                            Wild type                                                                            Mutant Y96A                                          ΔSpin                                                                            ΔSpin                                                                            NADH   NADH                  P450cam-substrate interactions high/    high/    turn-  turn-                 Subgroup: 3-ring               low Vs DTT                                                                             low Vs DTT                                                                             over?                                                                             GC?                                                                              over?                                                                             GC?               __________________________________________________________________________     ##STR50##        Anthracene                                                   ##STR51##        Phenanthrene --  --   20  20          +                      ##STR52##        Fluorene     --  --   --  50                                 ##STR53##        2-Fluorencarboxzaldehyde                                                                   --  --   --  50                                 ##STR54##        9-Fluorenone --  20   --   5                                 ##STR55##        Anthrone     --   5   --   5                                 ##STR56##        Anthraquinone                                                ##STR57##        2-Ethylanthraquinone                                        __________________________________________________________________________

                                      TABLE 2(e)                                  __________________________________________________________________________                                   Wild type                                                                              Mutant Y96A                                                                            Wild type                                                                            Mutant Y96A                                          ΔSpin                                                                            ΔSpin                                                                            NADH   NADH                  P450cam-substrate interactions high/    high/    turn-  turn-                 Subgroup: 4,5-ring             low Vs DTT                                                                             low Vs DTT                                                                             over?                                                                             GC?                                                                              over?                                                                             GC?               __________________________________________________________________________     ##STR58##        Chrysene     --  --   --  --                                 ##STR59##        1,2-Benzanthracene                                                                         --  --   --  --                                 ##STR60##        Fluoranthene --  5    20  10                                 ##STR61##        Pyrene*      --  --   --  --                                 ##STR62##        Perylene*    --  --   --  --                                __________________________________________________________________________

                                      TABLE 2(f)                                  __________________________________________________________________________                                   Wild type                                                                              Mutant Y96A                                                                            Wild type                                                                            Mutant Y96A                                          ΔSpin                                                                            ΔSpin                                                                            NADH   NADH                  P450cam-substrate interactions high/    high/    turn-  turn-                 Subgroup: Cyclic Alkanes       low Vs DTT                                                                             low Vs DTT                                                                             over?                                                                             GC?                                                                              over?                                                                             GC?               __________________________________________________________________________     ##STR63##        cis-Decahydro-  naphthalene                                                                nd  nd   nd  nd                                 ##STR64##        trans-Decahydro  naphthalene                                                               20  10   90  70                                 ##STR65##        Cyclohexane  --  --   60  60          +                      ##STR66##        Methylcyclohexane                                                                          50  50   100 70                                __________________________________________________________________________

                                      TABLE 2(g)                                  __________________________________________________________________________                                   Wild type                                                                              Mutant Y96A                                                                            Wild type                                                                            Mutant Y96A                                          ΔSpin                                                                            ΔSpin                                                                            NADH   NADH                  P450cam-substrate interactions high/    high/    turn-  turn-                 Subgroup: n-Alkanes            low Vs DTT                                                                             low Vs DTT                                                                             over?                                                                             GC?                                                                              over?                                                                             GC?               __________________________________________________________________________    n-Pentane                      --   5   55  40          +                     n-Hexane                       --  --   60  40          +                     n-Heptane                      5    5   60  40          +                     n-Octane                       --   5   80  45          +                     n-Nonane                       --  --   70  45          +                     n-Decane                       nd  nd   nd  nd                                n-Undecane                     nd  nd   20  20                                n-Dodecane                     nd  nd    5   5                                CH.sub.3 (CH.sub.2).sub.14 CH.sub.3                                                             n-Hexadecane --  --   --  --                                CH.sub.3 (CH.sub.2).sub.15 CH.sub.3                                                             n-Heptadecane                                                                              --  --   --  --                                CH.sub.3 (CH.sub.2).sub.11 OSO.sub.3,Na                                                         SDS          --  20   --  60                                CH.sub.3 (CH.sub.2).sub.7 CH═CH(CH.sub.2).sub.7 CO.sub.2 H                                  Oleic acid*  --   10? --   20?                              [(CH.sub.3).sub.2 CH(CH.sub.2).sub.3 CH(CH.sub.3)(CH.sub.2).sub.3             CH(CH.sub.3)CH.sub.2 CH.sub.2 --].sub.2                                                         Squalene     --  --   --  20                                 ##STR67##        Isoprene     --  --   10  10                                __________________________________________________________________________

                                      TABLE 2(h)                                  __________________________________________________________________________                           Wild type Mutant Y96A                                                                              Wild type                                                                              Mutant Y96A              P450cam-substrate interactions                                                                       ΔSpin                                                                             ΔSpin                                                                              NADH     NADH                     Subgroup: Camphor-like high/low                                                                           Vs DTT                                                                             high/low                                                                           Vs DTT                                                                              turnover?                                                                           GC?                                                                              turnover?                                                                           GC?                __________________________________________________________________________     ##STR68##                                                                              (1R)-(-)-Camphorquinone                                                                    80   80   80   80                                       ##STR69##                                                                              (1R)-(-)-Fenchone                                                                          40   70   50   80                                       ##STR70##                                                                              Dicyclopentadiene                                                                          50   80   90   90                                      __________________________________________________________________________

                  TABLE 3                                                         ______________________________________                                        Turnover of Small Alkanes by P450cam Mutants                                  All mutants listed below also contain the C334A mutation.                     Turnover rate measured as NADH consumption rate                               (nmole NADH/nmole P450cam/s).                                                 Alkane substrate:                                                             Main chain               Wild                                                 length   Name            type       Y96A                                      ______________________________________                                        C4       n-butane        --         --                                        C4       2-methyl butane background 4.6                                       C4       2,3-dimethyl butane                                                                           background 16.8                                      C4       2,2-dimethyl butane                                                                           background 14.0                                      C5       n-pentane       background 5.8                                       C5       2-methyl pentane                                                                              3.8        11.7                                      C5       3-methyl pentane                                                                              1.3        14.2                                      C5       2,4-dimethyl pentane                                                                          0.2        12.6                                      C5       2,2-dimethyl pentane                                                                          5.2        12.8                                      C5       2,2,4-trimethyl pentane                                                                       0.9        5.3                                       C5       3-ethyl pentane background 16.2                                      C6       n-hexane        background 6.0                                       C6       2-methyl hexane background 10.6                                      C7       n-heptane       2.7        4.4                                       C7       2-methyl heptane                                                                              background 2.1                                       C7       4-methyl heptane                                                                              1.4        10.2                                      C8       n-octane        background 5.8                                       C7       cycloheptane    4.4        42.5                                      ______________________________________                                         Product structures and distributions following oxidation of substrates        with P450cam active site mutants.                                             "background" - typical background NADH oxidation rate is 0.07 nmole NADH      (nmole P450cam).sup.-1 sec.sup.-1                                        

                                      TABLE 4(a)                                  __________________________________________________________________________    Product structure and distributions following oxidation of substrates         with P450cam                                                                  active site mutants. All mutants shown below also contain the C334A           mutation.                                                                     Cyclohexylbenzene      Products (%) for mutants:                              Products               WT Y96A                                                                              Y96F                                                                              Y96L                                                                              Y96V                                    __________________________________________________________________________     ##STR71##         3-ol                                                                              43 20  54  38  28                                       ##STR72##         3-ol                                                                              20 20  27  23  39                                       ##STR73##         Trans-  4-ol                                                                      25 15  6   23  10                                       ##STR74##         Cis-  4-ol                                                                        12 45  13  16  23                                      Total products (area/10.sup.5)                                                                       0.8                                                                              7.4 1.1 10.4                                                                              12.5                                     ##STR75##                                                                     ##STR76##                                                                     ##STR77##                                                                    __________________________________________________________________________

                  TABLE 4(b)                                                      ______________________________________                                                                Products (%) for                                      Phenylcyclohexene       mutants:                                              Products                WT       Y96A                                         ______________________________________                                         ##STR78##         3-one (A)                                                                              24       25                                        ##STR79##         3-ol (B) 76       75                                       Total products (area/10.sup.6)                                                                        42       36                                            ##STR80##                                                                     ##STR81##                                                                     ##STR82##                                                                    ______________________________________                                    

                                      TABLE 4(c)                                  __________________________________________________________________________                 Products (%) for mutants:                                        Naphthalene                         F87A-F96G-                                Products        WT  Y96A                                                                              Y96F                                                                              Y96L                                                                              Y96V                                                                              F193A                                     __________________________________________________________________________     ##STR83##   1-ol                                                                             100 100 100 100 100 100                                        ##STR84##   2-ol                                                                             0   0   0   0   0   0                                         Total products (area/10.sup.5)                                                                (0.016)                                                                           1.1 2.4 0.7 1.4 0.1                                        ##STR85##                                                                                                   ##STR86##                                      __________________________________________________________________________

                  TABLE 4(d)                                                      ______________________________________                                               Products (%) for mutants:                                                                                        F87A-                               Phenanthrene                              F96G-                               Products WT     Y96A    Y96F  Y96L  Y96V  F193A                               ______________________________________                                        A        38     49      41    35.5  41    27                                  B        15     23      31    41    38    41                                  C        12     13      5     9     11    3                                   D        35     15      23    14.5  10    29                                  Total products                                                                         0.075  7.0     4.5   2.8   1.6   0.065                               (area/10.sup.6)                                                                ##STR87##                                                                    ______________________________________                                    

                  TABLE 4(e)                                                      ______________________________________                                        Products (%) for mutants:                                                     Fluoranthene                             F87A-F96G-                           Products                                                                              WT     Y96A    Y96F  Y96L  Y96V  F193A                                ______________________________________                                        A       0      84      --    --    --    0                                    B       0      16      --    --    --    100                                  Total   0      2.7     --    --    --    0.2                                  products                                                                      (area/10.sup.6)                                                               ______________________________________                                         ##STR88##                                                                     -                                                                        

                  TABLE 4(f)                                                      ______________________________________                                        Products (%) for mutants:                                                     Pyrene                                   F87A-F96G-                           Products                                                                              WT     Y96A    Y96F  Y96L  Y96V  F193A                                ______________________________________                                        A       0      40      43    23    30    33                                   B       0      43.6    29    64.5  55    40                                   C       0      5       12.5  7.9   12    20                                   D       0      11.4    15.5  4.6   3     7                                    Total   0      1.2     1.5   1.5   1.6   0.02                                 products                                                                      (area/10.sup.6)                                                               ______________________________________                                         ##STR89##                                                                     -                                                                        

                  TABLE 4(g)                                                      ______________________________________                                        Lindane Products   Products (%) for mutants                                   (hexachlorocyclohexane)                                                                          WT       Y96A                                              ______________________________________                                        A                  100      100                                               Turnover rate      7.5      43.5                                              nmole NADH (nmolP450).sup.-1 s.sup.-1                                         ______________________________________                                         ##STR90##                                                                     -                                                                        

                  TABLE 4(h)                                                      ______________________________________                                                          Products (%)                                                                  for mutants:                                                                  Y96F Y96A                                                   ______________________________________                                        Hexane Products                                                               2-hexanone          10     15                                                 3-hexanone          16     28                                                 2-hexanol           24     26                                                 3-hexanol           50     32                                                 Relative activity   18.2   25.5                                               (WT = 1)                                                                      2-Methyl hexane Products                                                      2-methyl-2-hexanol  72     74                                                 5-methyl-2-hexanone 16     14                                                 2-methyl-3-hexanol  7      4                                                  5-methyl-2-hexanol  5      8                                                  Relative activity   2.3    2.6                                                (WT = 1)                                                                      ______________________________________                                    

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We claim:
 1. A mutant mono-oxygenase cytochrome P-450_(cam) wherein thetyrosine residue at position 96 is replaced by the residue of a smallhydrophobic amino acid.
 2. The mutant of claim 1, wherein said mutantcatalyzes the oxidation of a compound selected from the group consistingof a polycyclic aromatic hydrocarbon, a linear or branched alkane, abiphenyl compound and a halogenated hydrocarbon.
 3. The mutant of claim1, wherein the amino acid is selected from the group consisting ofalanine, glycine, isoleucine, leucine, and valine.
 4. The mutant ofclaim 1, wherein an amino acid residue at one or more of the positions87, 98, 185, 244, 247, 295 or 297 is independently replaced by anotheramino acid residue.
 5. The mutant of claim 2, wherein the amino acid isselected from the group consisting of alanine, glycine, isoleucine,leucine, and valine.
 6. The mutant of claim 2, wherein an amino acidresidue at one or more of the positions 87, 98, 185, 244, 247, 295 or297 is independently replaced by another amino acid residue.
 7. Themutant of claim 3, wherein an amino acid residue at one or more of thepositions 87, 98, 185, 244, 247, 295 or 297 is independently replaced byanother amino acid residue.
 8. A method of oxidizing a compound selectedfrom the group consisting of a polycyclic aromatic hydrocarbon, a linearor branched alkane, a biphenyl compound or a halogenated variant thereofand a halogenated hydrocarbon, comprising the step of contacting saidcompound under oxidizing conditions with mono-oxygenase cytochromeP-450_(cam) wherein the tyrosine residue at position 96 is replaced by asmall hydrophobic amino acid residue.
 9. The method of claim 8, whereinthe amino acid is selected from the group consisting of alanine,glycine, isoleucine, leucine, and valine.
 10. The method of claim 8,wherein an amino acid residue at one or more of the positions 87, 98,185, 244, 247, 295 or 297 is independently replaced by another aminoacid residue.
 11. The method of claim 9, wherein an amino acid residueat one or more of the positions 87, 98, 185, 244, 247, 295 or 297 isindependently replaced by another amino acid residue.